The objective of this mechanism-based research is to devise a safe and efficient, synthetic lipid- based gene carrier for use in parenteral gene therapy in animals. We postulate that molecular strategies to deliver nucleic acids by viral membrane fusion proteins can be mimicked using less complex lipids. We will design and synthesize lipids that mediate a triggered-membrane fusion and assemble them with plasmid DNA to create a sub-100 nm, nanolipid particle (NLP) that mediates high transfection with low toxicity in animals. The research plan contains four specific aims. Specific aim 1: To design, synthesize and characterize novel lipids that alter their charge, interfacial hydrophobicity and phase preference at low pH. The design explores the effect of systematic alterations in the chemical structure of the head group, linker region and hydrophobic portion of the lipids on liposome collapse at low pH. Specific aim 2: To formulate lipids synthesized in aim 1 so that they undergo a low pH triggered lamellar to a hexagonal phase change and to characterize the formulations to confirm their phase preference. Specific aim 3: To measure the transfection activity and cytotoxicity of NLP formulations encapsulating luciferase encoding plasmid DNA as a function of the amount of lipid that binds to the cell. To measure the rate, location and extent of cytoplasmic contents delivery using fluorescence confocal microscopy and correlate this with the pH of the compartment when delivery is first observed. To test the hypothesis that incorporation of an appropriate targeting ligand or membrane destabilizing peptide can further increase gene transfer. Specific aim 4: To characterize the extent and duration of gene transfer in liver hepatocytes or in a flank tumor in mice as a function of NLP composition after a low volume intravenous dose of an optimized targeted NLP. Completion of these specific aims will result in a 'lipid engineered' synthetic gene carrier with an in vivo gene transfer efficiency approaching that of current viral vectors. The resulting NLP will be a non- inflammatory vector in circulation and mediate rapid cytoplasmic transfer of nucleic acids after internalization into cells. Success in this research will enable safe and effective gene therapy for a variety of currently untreatable genetic disorders and neoplastic diseases.